KR20220017946A - Anti-B7-H4 antibody-drug conjugates and medical uses thereof - Google Patents

Abstract

Anti-B7-H4 antibody-drug conjugates and medical uses thereof are provided. Specifically, an anti-B7-H4 antibody or antigen-binding fragment thereof, a humanized antibody comprising the CDR regions of the anti-B7-H4 antibody, and an antibody-drug conjugate thereof or a pharmaceutically acceptable salt or solvate thereof , and pharmaceutical compositions of said antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof, and their use as anticancer drugs, in particular in the manufacture of drugs for the treatment of diseases or disorders having high expression of B7-H4 provide use.

Description

Anti-B7-H4 antibody-drug conjugates and medical uses thereof

The present invention relates to the field of biomedical science. Specifically, the present invention relates to anti-B7-H4 antibody-drug conjugates and medical uses thereof.

Tumor immunotherapy is a long-term sustainable research and development hotspot in the field of oncology, and T-cell tumor immunotherapy is at its core. Tumor avoidance is a major hurdle for tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees. However, in many cases inefficient activation of effector T cells results in an insufficient immune response, and thus tumor cells promote tumor growth by virtue of their inhibitory effect on the immune system. Tumor immunotherapy is the maximization and recruitment of killer T cells and/or other immune cells from a patient with a tumor to kill the tumor.

Studies of the CD28 receptor and its ligands have led to the characterization of molecules called the B7 superfamily. Members of the B7 family are a class of immunoglobulins having an immunoglobulin-like V domain (IgV) and an immunoglobulin-like C domain (IgC). Its members are co-stimulatory factors B7.1 (CD80) and B7.2 (CD86), inducible co-stimulatory factor ligands (ICOS-L/B7-H2), programmed cell death-1 ligands (PD-L1/B7- H1), programmed cell death-2 ligand (PD-L2/B7-DC), B7-H3 and B7-H4, and the like.

Human B7-H4 is a type I transmembrane protein of 282 amino acids. Its coding gene is located in the p11.1 region of chromosome 1 (Choi IH et al., J Immunol. 2003 Nov 1; 171(9): 4650-4). B7-H4 has a negative regulatory effect on the immune response of T cells. B7-H4 plays a broad inhibitory role in the differentiation and development of CD4+ and CD8+ T cells, cell cycle progression and cytokine production (Sica GL et al., Immunity. 2003 Jun; 18(6): 849-61). ). No immune cell dysfunction or autoimmune events were found in B7-H4 knockout mice (Zhu G et al., Blood. 2009 Feb 19; 113(8): 1759-67; Suh WK et al. , Blood. Mol Cell Biol. 2006 Sep; 26(17): 6403-11]). Currently, the receptors of B7-H4 and its signaling pathway are still unclear.

Recent studies have found that the B7-H4 protein is abundantly expressed in various tumor tissues, which enables tumor cells to evade attack by the body's immune system. Considering the B7-H4 molecule as a target for tumor therapy provides a novel approach to tumor immunotherapy.

Currently, human B7-H4 is known to be expressed on cancer cells such as breast cancer, ovarian cancer, lung cancer, cervical cancer, kidney cancer, bladder cancer and liver cancer. Expression of B7-H4 mRNA is found in the spleen, lung, thymus, liver, skeletal muscle, kidney, pancreas, testis and ovary. At the protein level, B7-H4 expression is found at low levels in tissues such as the breast (ducts and lobules), fallopian tube epithelium, and endometrial glands. Related studies have also shown that B7-H4 is overexpressed in tumor-associated macrophages (TAM) (Kryczek, I. et al., J. Exp. Med. 2006, 203(4): 871-881), versus It has been demonstrated that phagocytes constitute an important component of the tumor microenvironment and can account for as much as 50% of the tumor mass.

One strategy for cancer treatment is to use antibodies as carriers to deliver cytotoxic molecules to cancer cells and then kill the cancer cells by dissociated small molecules. The drugs used in this strategy are called antibody-drug conjugates. ADCetris and Kadcyla are currently marketed antibody-drug conjugates. Currently, many multinational pharmaceutical companies are developing monoclonal antibodies against B7-H4 and/or its drug conjugates to improve the patient's immune system response to tumors and to achieve the goal of directly killing tumor cells. Relevant patents are, for example, WO20133025779, US20140322129 and the like. Anti-B7-H4 monoclonal antibodies from Medimmune, FivePrime and others are currently still in preclinical development; Genentech's anti-B7-H4 antibody-drug conjugate is also in preclinical development.

An object of the present invention is to provide an anti-B7-H4 antibody-drug conjugate having high affinity, high selectivity, high phagocytosis efficiency, high anticancer activity, high stability, high safety, and low toxicity and side effects, which is achieved through the following technical solution will provide

An antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof:

[Formula A]

In the above formula,

D is a cytotoxic drug;

L ₁ and L ₂ are linker units;

y is a number from 1 to 20;

Ab is a B7-H4 antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region;

wherein said antibody heavy chain variable region comprises one or more HCDRs set forth in a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11;

The antibody light chain variable region comprises one or more LCDRs set forth in a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

In one embodiment of the present invention, the antibody heavy chain variable region also comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31; or

said antibody heavy chain variable region may also comprise one or more HCDRs set forth in a sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45;

The antibody light chain variable region comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 33 and one or more LCDRs set forth in a sequence selected from the group consisting of SEQ ID NO:34.

In one embodiment of the present invention, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the anti-B7-H4 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region wherein the antibody heavy chain variable region comprises

HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5; or

HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11

includes

In one embodiment of the present invention, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the anti-B7-H4 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region wherein the antibody heavy chain variable region comprises

HCDR1 shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24 and HCDR3 shown in SEQ ID NO:25; or

HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31

includes

In one embodiment of the present invention, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the anti-B7-H4 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region wherein the antibody heavy chain variable region comprises

HCDR1 set forth in SEQ ID NO: 43, HCDR2 set forth in SEQ ID NO: 44 and HCDR3 set forth in SEQ ID NO: 25; or

HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 45 and HCDR3 set forth in SEQ ID NO: 31

includes

In one embodiment of the present invention, in the antibody-drug conjugate of Formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the antibody heavy chain variable region of Ab consists of the following (1) to (4) comprising any one CDR selected from the group:

(1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5;

(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11;

(3) HCDR1 set forth in SEQ ID NO:23, HCDR2 set forth in SEQ ID NO:24 and HCDR3 set forth in SEQ ID NO:25; and

(4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31.

The antibody heavy chain variable region of said Ab may also comprise a CDR selected from the group consisting of (5) to (6):

(5) HCDR1 set forth in SEQ ID NO:43, HCDR2 set forth in SEQ ID NO:44 and HCDR3 set forth in SEQ ID NO:25; and

(6) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 45 and HCDR3 set forth in SEQ ID NO: 31.

In one embodiment of the present invention, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the anti-B7-H4 antibody or antigen-binding fragment thereof comprises an antibody light chain variable region wherein the antibody light chain variable region comprises

LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively

includes

In some embodiments of the present invention, in the antibody-drug conjugate of Formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the anti-B7-H4 antibody or antigen-binding fragment thereof comprises an antibody light chain variable region wherein said antibody light chain variable region comprises

LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively; or

LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively

includes

In one embodiment of the present invention, in the antibody-drug conjugate of Formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the antibody heavy chain variable region of Ab consists of the following (1) to (4) comprising any one CDR selected from the group:

(1) LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8;

(2) LCDR1 set forth in SEQ ID NO: 12, LCDR2 set forth in SEQ ID NO: 13 and LCDR3 set forth in SEQ ID NO: 14;

(3) LCDR1 set forth in SEQ ID NO:26, LCDR2 set forth in SEQ ID NO:27 and LCDR3 set forth in SEQ ID NO:28; and

(4) LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34.

In one embodiment of the present invention, in the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, the heavy and light chains of the anti-B7-H4 antibody or antigen-binding fragment thereof are variable area is

(1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5, respectively; or

LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7 and LCDR3 set forth in SEQ ID NO: 8; or

(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11, respectively; or

LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13 and LCDR3 shown in SEQ ID NO: 14

includes

In one embodiment of the present invention, in the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, the heavy and light chains of the anti-B7-H4 antibody or antigen-binding fragment thereof are variable area is

(3) HCDR1 set forth in SEQ ID NO:23, HCDR2 set forth in SEQ ID NO:24 and HCDR3 set forth in SEQ ID NO:25; or

LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28; or

(4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31; or

LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34

includes

In one embodiment of the present invention, in the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, the heavy and light chains of the anti-B7-H4 antibody or antigen-binding fragment thereof are variable area is

(5) HCDR1 set forth in SEQ ID NO: 43, HCDR2 set forth in SEQ ID NO: 44 and HCDR3 set forth in SEQ ID NO: 25, respectively; or

LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28; or

(6) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 45 and HCDR3 set forth in SEQ ID NO: 31, respectively; or

LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34

includes

In one embodiment of the present invention, in the antibody-drug conjugate of Formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the antibody heavy chain variable region and the antibody light chain variable region of Ab are selected from the following (1) to (4) comprises any one CDR selected from the group consisting of:

(1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5; and

LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7 and LCDR3 set forth in SEQ ID NO: 8;

(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11; and

LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13 and LCDR3 shown in SEQ ID NO: 14;

(3) HCDR1 set forth in SEQ ID NO:23, HCDR2 set forth in SEQ ID NO:24 and HCDR3 set forth in SEQ ID NO:25; and

LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28; and

(4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31; and

LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34.

The antibody heavy chain variable region and antibody light chain variable region of said Ab may also comprise a CDR selected from the group consisting of (5) or (6):

(5) HCDR1 set forth in SEQ ID NO:43, HCDR2 set forth in SEQ ID NO:44 and HCDR3 set forth in SEQ ID NO:25; and

LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28;

(6) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 45 and HCDR3 set forth in SEQ ID NO: 31; and

LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, and A humanized antibody or fragment thereof.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab further comprises a light chain framework region and a heavy chain framework region sequence, each of which is a human derived from germline light and heavy chain sequences or mutant sequences thereof;

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab further comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG1 or a variant thereof. , IgG2 or variants thereof, IgG3 or variants thereof, or regions derived from IgG4 or variants thereof, preferably regions derived from human IgG1, IgG2 or IgG4, more preferably IgG1 heavy chain with enhanced ADCC toxicity after amino acid mutation a constant region, most preferably a heavy chain constant region set forth in SEQ ID NO:54;

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is a light chain constant region derived from a human κ chain, a λ chain or a variant thereof, preferably It further comprises said region derived from a human κ chain, more preferably a light chain constant region set forth in SEQ ID NO:55.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab comprises a light chain variable region having the following sequence: SEQ ID NO: 16 or SEQ ID NO: 18 .

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is of the following sequence: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36 or SEQ ID NO: 38 a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36 or SEQ ID NO: 38 variable regions.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab comprises a heavy chain variable region having the following sequence: SEQ ID NO: 15 or SEQ ID NO: 17 .

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is of the following sequence: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 37 a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 37 variable regions.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the heavy chain variable region and the light chain variable region of Ab are any one selected from the group consisting of:

(1) a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16;

(2) a heavy chain variable region set forth in SEQ ID NO: 17 and a light chain variable region set forth in SEQ ID NO: 18.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the heavy chain variable region and the light chain variable region of Ab are any one selected from the group consisting of:

(1) a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16;

(2) a heavy chain variable region set forth in SEQ ID NO: 17 and a light chain variable region set forth in SEQ ID NO: 18;

(3) a heavy chain variable region set forth in SEQ ID NO: 35 and a light chain variable region set forth in SEQ ID NO: 36; and

(4) a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 38.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab comprises a light chain variable region having the following sequence: SEQ ID NO: 20 or SEQ ID NO: 22 .

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is of the following sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 40 or SEQ ID NO: 42 or a full-length light chain having at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 20, or SEQ ID NO: 22, SEQ ID NO: 40 or SEQ ID NO: 42.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab comprises a heavy chain having the following sequence: SEQ ID NO: 19 or SEQ ID NO: 21.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab comprises the following sequences: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 39, SEQ ID NO: 41 or a full-length heavy chain having at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 39, SEQ ID NO: 41.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is

(1) a light chain of SEQ ID NO: 20 and a heavy chain of SEQ ID NO: 19; or

(2) the light chain of SEQ ID NO: 22 and the heavy chain of SEQ ID NO: 21

includes

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, Ab is

(1) a light chain of SEQ ID NO: 20 and a heavy chain of SEQ ID NO: 19; or

(2) a light chain of SEQ ID NO: 22 and a heavy chain of SEQ ID NO: 21; or

(3) a light chain of SEQ ID NO: 40 and a heavy chain of SEQ ID NO: 39; or

(4) the light chain of SEQ ID NO: 42 and the heavy chain of SEQ ID NO: 41

includes

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the antigen-binding fragment of the anti-B7-H4 antibody is Fab, Fab', F(ab' ) ₂ , an antigen-binding fragment of a peptide comprising a single chain antibody (scFv), a dimerized V region (diabody), a disulfide bond stabilized V region (dsFv) and a CDR.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the cytotoxic drug is a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope and a nuclease; Preferably, a tubulin inhibitor or a DNA topoisomerase inhibitor that inhibits cell division; more preferably DM1, DM3, DM4, SN-38, MMAF and MMAE; more preferably selected from the group consisting of the tubulin inhibitors SN-38, MMAE and MMAF. Here, the structures of MMAF and SN-38 are as shown in the following equations:

.

.

In a preferred embodiment, in the antibody-drug conjugate of formula A as described above, or a pharmaceutically acceptable salt or solvate thereof, the cytotoxic drug is selected from camptothecin derivatives, preferably Exatecan:

.

.

In a preferred embodiment, the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above is a compound of formula (I): or a pharmaceutically acceptable salt or solvate thereof:

[Formula I]

In the above formula,

L ₁ and L ₂ are linker units;

y is a number selected from 1 to 8, preferably a number selected from 2 to 4;

Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

In a preferred embodiment, the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of formula (II), or a pharmaceutically acceptable salt or solvate thereof:

[Formula II]

In the above formula,

L ₁ and L ₂ are linker units;

y is a number selected from 1 to 8, preferably a number selected from 2 to 4;

Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

In a preferred embodiment, the antibody-drug conjugate of formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of formula (III), or a pharmaceutically acceptable salt or solvate thereof:

[Formula III]

In the above formula,

L ₁ and L ₂ are linker units;

y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8;

y is a number selected from 2 to 10, more preferably a number selected from 6 to 10, more preferably a number from 7 to 9, most preferably an integer from 7, 8, 9;

Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

In a preferred embodiment, in the antibody-drug conjugate as described above, or a pharmaceutically acceptable salt or solvate thereof, L ₁ is as shown in Formula B:

[Formula B]

In the above formula,

M ₁ is —CR ₁ R ₂ —;

R ₁ and R ₂ are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino;

N is an integer from 0 to 5, preferably 1, 2 or 3.

In a preferred embodiment, in the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, L ₂ is as shown in Formula (C):

[Formula C]

In the above formula,

M ₂ is —CR ₄ R ₅ —;

R ₃ is selected from the group consisting of a hydrogen atom, halogen, hydroxyl, amino, alkyl, alkoxyl and cycloalkyl;

R ₄ and R ₅ are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino;

m is an integer from 0 to 5, preferably 1, 2 or 3.

In a preferred embodiment, in the antibody-drug conjugate as described above, or a pharmaceutically acceptable salt or solvate thereof, L ₂ is as shown in Formula D:

[Formula D]

-K ¹ -K ² -K ³ -K ⁴ -

In the above formula,

이고, s는 2 내지 8의 정수, 더욱 바람직하게는 4 내지 8의 정수, 보다 바람직하게는 4 내지 6의 정수이고;K ¹ is

, s is an integer from 2 to 8, more preferably an integer from 4 to 8, more preferably an integer from 4 to 6;

K ² is -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ CH ₂ C(O)-, -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ C(O)-, -S(CH ₂ ) _p C(O)- or a single bond, p is an integer from 1 to 20, preferably from 1 to 6;

R ₁ is selected from the group consisting of hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;

K ³ is a tetrapeptide residue, preferably the tetrapeptide residue is a peptide residue formed by an amino acid selected from the group consisting of at least two of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate and aspartate; more preferably the tetrapeptide residue GGFG;

K ⁴ is —NR ² (CR ³ R ⁴ )t—, and R ² , R ³ and R ⁴ are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydride. hydroxyalkyl; t is 1 or 2,

preferably with respect to L ₂ ,

이고, s는 5이고;K ¹ is

and s is 5;

K ² is a bond;

K ³ is the tetrapeptide residue GGFG;

K ⁴ is —NR ² (CR ³ R ⁴ )t-, and R ² , R ³ and R ⁴ are each independently hydrogen, deuterium, hydroxyl, amino, C _1-6 alkyl, halogen, C _1-6 halo alkyl, C _1-6 deuterated alkyl and C _1-6 hydroxyalkyl, and t is 1 or 2.

In a preferred embodiment, in the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof, the K ¹ terminus of the linker unit -L ₂ - is linked to Ab, and the K ⁴ terminus is linked to L ₁ . do.

In a preferred embodiment, in the antibody-drug conjugate as described above, or a pharmaceutically acceptable salt or solvate thereof, L ₁ is -O-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)- , -O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -, -O-CR ⁵ R ⁶ -, -NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)- or - S-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-;

R ^a and R ^b are each independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl;

R ⁵ is haloalkyl or cycloalkyl,

R ⁶ is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl,

R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl;

m is 0, 1, 2, 3 or 4.

Preferably, the O terminus of L ₁ is connected to the linker unit L ₂ .

In a preferred embodiment, in the antibody-drug conjugate as described above, or a pharmaceutically acceptable salt or solvate thereof, L ₁ is as shown in Formula E:

[Formula E]

In the above formula,

R ⁵ is haloalkyl or cycloalkyl,

R ⁶ is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl,

R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl;

preferably

R ⁵ is selected from the group consisting of C _1-6 haloalkyl and C _3-6 cycloalkyl,

R ⁶ is selected from the group consisting of hydrogen, C _1-6 haloalkyl and cycloalkyl,

R ⁵ and R ⁶ and the carbon atom to which they are connected form a C _3-6 cycloalkyl;

m is an integer from 0 to 4;

More preferably, formula E is selected from the group consisting of the following substituents:

In a more preferred embodiment, in the antibody-drug conjugate as described above, or a pharmaceutically acceptable salt or solvate thereof, -L ₂ -L ₁ - is of the structure:

In the above formula,

K ² is a bond;

K ³ is the tetrapeptide residue GGFG;

R ⁵ is haloalkyl or C _3-6 cycloalkyl,

R ⁶ is selected from the group consisting of hydrogen, haloalkyl and C _3-6 cycloalkyl,

R ⁵ and R ⁶ and the carbon atom to which they are connected form a C _3-6 cycloalkyl;

R ² , R ³ and R ⁴ are each independently hydrogen or alkyl;

s is an integer from 2 to 8; Preferably, s is 4, 5 or 6;

m is an integer from 0 to 4;

Preferably, said -L ₂ -L ₁ - is selected from the group consisting of:

In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in Formula IV:

[Formula IV]

In the above formula,

W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-cycloalkyl or linear heteroalkyl of 1 to 8 atoms, wherein said heteroalkyl is 1 to 3 selected from the group consisting of N, O or S heteroatoms, wherein each of said C _1-8 alkyl, cycloalkyl or linear heteroalkyl is independently halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl optionally further substituted with one or more substituents selected from the group consisting of;

K ² is -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, -S(CH ₂ ) _p1 C(O)— and a bond, R ¹ is selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, p ₁ is an integer from 1 to 20;

K ³ is a peptide residue consisting of 2 to 7 amino acids, which may be substituted or unsubstituted. When substituted, said substituent may be substituted at any available point of attachment, said substituent being from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl. at least one independently selected from;

R ² is independently selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ³ and R ⁴ are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁵ is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R ⁶ is selected from the group consisting of a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl or heterocyclyl;

m is an integer from 0 to 4;

y is 1 to 10 and y is a prime number or integer, preferably y is a number from 2 to 10, more preferably y is a number from 4 to 10, more preferably a number from 6 to 9, most preferably is an integer of 7, 8 or 9;

Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof.

In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of Formula I-A, or a pharmaceutically acceptable salt or solvate thereof:

[Formula I-A]

In the above formula,

y is 1 to 10, and y is a decimal or integer.

In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of formula I-B, or a pharmaceutically acceptable salt or solvate thereof:

[Formula I-B]

In the above formula,

y is 1 to 10, and y is a decimal or integer.

In a more preferred embodiment, the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof is an antibody-drug conjugate of Formula II-A, or a pharmaceutically acceptable salt or solvate thereof:

[Formula II-A]

In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of Formula II-B, or a pharmaceutically acceptable salt or solvate thereof:

[Formula II-B]

In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above is an antibody-drug conjugate of Formula IV-A, or a pharmaceutically acceptable salt or solvate thereof:

[Formula IV-A]

In the above formula,

s is an integer from 2 to 8, R ² to R ⁶ , m and y are as defined in Formula IV above;

Preferably, s is an integer of 4, 5 or 6, y is a number from 4 to 10, preferably a number from 6 to 9, and more preferably 7 or 8.

In a more preferred embodiment, the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof is selected from the group consisting of:

In a most preferred embodiment, the antibody-drug conjugate as described above or a pharmaceutically acceptable salt or solvate thereof is selected from the group consisting of:

In the above formula,

y is a number selected from 1 to 10, preferably a number selected from 2 to 10, further a number selected from 6 to 10, more preferably a number from 7 to 9, most preferably 7, 8, 9 is an integer of; y is a number selected from 2 to 10, preferably a number from 4 to 8, more preferably a number from 6 to 8, even more preferably a number from 7 to 8, most preferably 8.

In a preferred embodiment, the present invention relates to a method for preparing an antibody-drug conjugate represented by formula (IV), or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of: The compound shown in Formula IV is prepared by performing a coupling reaction with the compound shown:

In the above formula,

Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof;

W, K ² , K ³ , R ² to R ⁶ , m and y are as defined in formula IV.

In a preferred embodiment, the compound represented by formula F is the compound represented by formula F-1 It is salt:

[Formula F-1]

In the above formula,

K ² , K ³ , R ² to R ⁶ , s and m are as defined in -L ₂ -L ₁ - above.

In a preferred embodiment, the compound represented by Formula F or Formula F-1 is selected from the group consisting of:

.

.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate according to any one of the present invention or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers. it's about

In a preferred embodiment, the present invention relates to an antibody-drug conjugate of formula (A) or a pharmaceutical thereof in the manufacture of a medicament for the treatment of a disease associated with human B7-H4, preferably a medicament for the treatment of a cancer having high B7-H4 expression. to the use of a chemically acceptable salt or solvate, or a pharmaceutical composition thereof.

In a more preferred embodiment, the cancer is astroblastoma of the human brain, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body Tumor, cervical cancer, chondrosarcoma, chordoma, chromophore cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative small cell tumor, ependymoma, Ewing's sarcoma, extraosseous mucinous chondrosarcoma , osseous incomplete fibrosis, fibrous dysplasia of bone, gallbladder or cholangiocarcinoma, gastric cancer, gestational villous disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, renal cancer, leukemia, liposarcoma/malignancy Lipoma tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, childhood cancer, peripheral schwannoma , pheocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis, rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thyroid metastasis and uterine cancer.

Figure 1: In vivo efficacy of antibody-drug conjugates: hu2G6-MC-MMAF and hu2F7-MC-MMAF showed inhibitory and killing effects on MX-1 xenograft tumors at doses of 1.5 mg/kg and 3 mg/kg it was
Figure 2: HPLC spectra of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively.
Figure 3: In vivo anti-tumor efficacy of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively.
Figure 4: In vivo anti-tumor efficacy of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively. The figure depicts the tumors of mice in each group at day 21.

1. Terminology

In order to better understand the present invention, some technical and scientific terms are specifically defined below. Unless explicitly defined otherwise in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter codes and one-letter codes of amino acids used in the present invention are described in J. Biol. Chem, 243, p3558 (1968)].

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by an interchain disulfide bond. The composition and order of amino acids in the immunoglobulin heavy chain constant region are different, and therefore their antigenicity is also different. Accordingly, immunoglobulins can be classified into five types (also known as isotypes of immunoglobulins): IgM, IgD, IgG, IgA and IgE, whose corresponding heavy chains are each μ chain, δ chain, γ chain, α chain and ε chain. Igs of the same type can be classified into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified as IgG1, IgG2, IgG3 and IgG4. Light chains are classified into κ chains or λ chains according to differences in constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region, wherein the region comprises a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, wherein the region comprises human or murine IgG1, 2, 3, 4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains is highly variable and is a variable region (V region); The remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. These three hypervariable regions determine the specificity of an antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxyl terminus. are arranged as the three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; The three CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The number and position of CDR amino acid residues in the VL region and VH region of the antibody or antigen-binding fragment of the present invention is determined by the known Kabat or Chothia numbering standards and Kabat or AbM definition standards (http://bioinf). .org.uk/abs/).

The term "antigen presenting cell" or "APC" is a cell presenting a foreign antigen that forms a complex with MHC on its surface. T cells use the T cell receptor (TCR) to recognize such complexes. Examples of APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), mononuclear cells, B lymphoblasts, and monocyte-derived dendritic cells (DC). The term "antigen presentation" refers to the process by which APCs capture an antigen and make it available for recognition by T cells, for example as a component of an MHC-I/MHC-II conjugate.

The term “B7-H4” refers to a member of the human B7 protein family (also known as CD276), which is a type I transmembrane protein having four Ig-like extracellular domains. B7-H4 is one of the immune checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells, and has an inhibitory effect on the functional activation of T cells. The term "B7-H4" includes any variant or isoform of B7-H4 that is naturally expressed by a cell. Antibodies of the invention are capable of cross-reacting with B7-H4 derived from a non-human species. As another option, the antibody may also be specific for human B7-H4 and not show cross-reactivity with other species. B7-H4 or any variant or isoform thereof can be isolated from cells or tissues that naturally express it, or produced by recombinant techniques using techniques commonly used in the art and techniques described herein. Preferably, said anti-B7-H4 antibody targets human B7-H4 with a conventional glycosylation pattern.

The term "recombinant human antibody" refers to a human antibody prepared, expressed, generated or isolated by recombinant methods, eg, (1) a transgenic or transchromosomal animal (eg, a mouse) expressing a human immunoglobulin gene. an antibody isolated from, or a hybridoma prepared therefrom; (2) an antibody isolated from a host cell transformed to express the antibody, eg, a transfected species; (3) an antibody isolated from a recombinant human antibody library; and (4) antibodies prepared, expressed, generated or isolated by splicing human immunoglobulin gene sequences with other DNA sequences and by other methods, related techniques and methods are well known in the art. Such recombinant human antibodies utilize specific human germline immunoglobulin sequences encoded by germline genes, but contain variable and constant regions that also contain subsequent rearrangements and mutations, such as those that occur during antibody maturation. include

In the present invention, the term "murine antibody" refers to a monoclonal antibody against human B7-H4 prepared according to the knowledge and technology in the art. During manufacture, test subjects are injected with the B7-H4 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are then isolated. In a preferred embodiment of the present invention, the murine B7-H4 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine κ, λ chain or a variant thereof, or of a murine IgG1, IgG2, IgG3 or IgG4 or variant thereof. It may further comprise a heavy chain constant region.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the invention contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). may include However, the term "human antibody" does not include antibodies (ie "humanized antibodies") in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences. does not

The term "humanized antibody" (also known as a CDR-grafted antibody) refers to an antibody generated by grafting murine CDR sequences into the framework of a human antibody variable region. Such antibodies are able to overcome the strong immune response induced by chimeric antibodies carrying large amounts of murine protein components. To avoid a decrease in activity caused by reduced immunogenicity, minimal back mutations can be made to the human antibody variable region to maintain this activity.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which is capable of ameliorating the immune response induced by the murine antibody. The establishment of a chimeric antibody is first to establish a hybridoma secreting a murine-specific monoclonal antibody, followed by cloning the variable region gene from the murine hybridoma cell, followed by cloning the constant region gene of the human antibody as necessary. , linking the murine variable region gene and the human constant region gene to form a chimeric gene (inserted into a human expression vector), and finally expressing the chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. . wherein said human antibody constant region preferably comprises a human IgG2 or IgG4 heavy chain constant region or uses an IgG1 with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation, human IgG1, heavy chain constant region of IgG2, IgG3 or IgG4 or variants thereof.

The term "antigen-binding fragment" refers to antigen-binding fragments of an antibody and antibody analogs, usually comprising at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of a parent antibody. The antibody fragment retains at least some of the binding specificities of the parent antibody. Generally, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are described in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136].

A “Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The “Fc” region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by at least two disulfide bonds and through hydrophobic interactions of the CH3 domain.

A "Fab' fragment" is an F comprising a light chain and a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains (thus, an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments). (ab')2 forms a molecule.

An “F(ab′)2 fragment” comprises two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

An “Fv region” includes variable regions from both heavy and light chains, but no constant regions.

The term "multispecific antibody" is used in its broadest sense and includes antibodies having multiepitope specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or to a different epitope on the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or to a different epitope on the same target; full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments linked together covalently or non-covalently, and the like.

The term "single chain antibody" is a single chain recombinant protein formed by linking the heavy chain variable region (VH) and light chain variable region (VL) of an antibody via a linker peptide. Such antibodies are the smallest antibody fragments with a complete antigen binding site.

The term "domain antibody fragment" is an immunoglobulin fragment with immunological function comprising only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same or different antigens.

As used herein, the term "binding to B7-H4" refers to the ability to interact with human B7-H4. The term "antigen-binding site" of the present invention refers to a three-dimensional site interspersed on an antigen and recognized by an antibody or antigen-binding fragment of the present invention.

As used herein, the terms “specifically binds” and “selectively binds” refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when recombinant human B7-H4 is used as an analyte and an antibody is used as a ligand, the antibody binds to the predetermined antigen at about 10 ^-7 M, as measured by surface plasmon resonance (SPR) technology in the instrument. binds with an equilibrium dissociation constant (KD) that is less than or even less than this, and its binding affinity for said predetermined antigen is for a non-specific antigen (other than said predetermined antigen or closely related antigen, e.g., BSA, etc.) at least twice its binding affinity. The term "antibody recognizing antigen" may be used herein interchangeably with the term "antibody that specifically binds to...".

The term “cross-reactivity” refers to the ability of an antibody of the invention to bind B7-H4 from different species. For example, an antibody of the invention that binds to human B7-H4 may also bind to B7-H4 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or by binding or functional interaction with cells physiologically expressing B7-H4. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) assays or flow cytometry.

The terms “inhibition” or “blocking” may be used interchangeably and include both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the level or type of activity normally present if ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of a ligand when contacted with an anti-B7-H4 antibody compared to the binding affinity of a ligand not contacted with the anti-B7-H4 antibody.

The term "inhibition of growth" (eg, when referring to cells) is intended to include any measurable decrease in cell growth.

The terms "induced immune response" and "enhanced immune response" are used interchangeably and refer to an immune response (ie, passive or adaptive) to stimulation by a particular antigen. The term "induce" as used in the expression "induction of CDC or ADCC" refers to stimulating a particular direct mechanism of apoptosis.

In the present invention, "ADCC", that is, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill target cells coated with the antibody by recognizing the Fc fragment of the antibody. The ADCC effector function of the antibody can be enhanced, reduced or abrogated by modifying the Fc segment of IgG. Such modifications refer to mutations in the heavy chain constant region of an antibody.

Methods for the production and purification of antibodies and antigen-binding fragments are well known and can be found in the prior art, for example, in Antibodies: A Laboratory Manual, Cold Spring Harbor, chapters 5-8 and 15. For example, mice can be immunized with human B7-H4 or a fragment thereof, the obtained antibody can be regenerated and restored, and amino acid sequencing can be performed using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. Antibodies or antigen-binding fragments of the invention are genetically engineered to introduce one or more human FR regions onto non-human CDR regions. Human FR germline sequences can be obtained from the ImmunoGeneTics (IMGT) website at http://imgt.cines.fr, or from Immunoglobulin FactsBook, 2001ISBN012441351.

The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can induce glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to a human antigen. Positive clones are expanded in serum-free medium in a bioreactor to generate antibodies. The culture medium in which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can also be removed by conventional methods, for example molecular sieves and ion exchange. The resulting product needs to be frozen or lyophilized immediately, for example at -70°C.

Antibodies of the invention refer to monoclonal antibodies. A monoclonal antibody (mAb) as described herein refers to an antibody obtained from a single cloned cell line, said cell line being, but not limited to, a eukaryotic, prokaryotic or phage cloned cell line. Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly, for example, using hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques (eg CDR-grafting) or other conventional techniques.

"Administering" and "treating" refer to an exogenous agent, therapeutic agent, diagnostic agent, or composition when applied to an animal, human, test subject, cell, tissue, organ, or biological fluid, said animal, human, subject, cell, tissue, organ. or contact with a biological fluid. “Administering” and “treating” may refer to, for example, treatment, pharmacokinetics, diagnostics, research and laboratory methods. Treating a cell comprises contacting a reagent with the cell, and contacting a reagent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean treating a cell in vitro and ex vivo, for example with a reagent, diagnostic agent, binding composition, or another type of cell. "Treating" refers to therapeutic treatment, prophylactic or prophylactic means, research and diagnostic applications when applied to human, veterinary or research subjects.

"Treatment" refers to administration of a composition comprising either an internal or external therapeutic agent, e.g., a binding compound of the present invention, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. . In general, the therapeutic agent is administered to alleviate one or more symptoms of a disease, induce regression of such symptoms, or inhibit the development of any clinically measurable degree of such symptoms in the treated subject or population. provided in an effective amount. The amount of a therapeutic agent effective to ameliorate any particular disease symptom (also referred to as a "therapeutically effective amount") depends on a variety of factors, including the disease state, age and weight of the patient, and the drug that produces the desired therapeutic effect in the patient. may vary depending on the ability of Whether a disease symptom has been alleviated can be assessed by any clinical test method commonly used by a physician or other medical professional for evaluation of the severity or progression of the symptom. Although embodiments of the present invention (eg, methods or products of treatment) may not be effective in alleviating each symptom of a disease of interest, these embodiments can be used to evaluate the symptom of a disease of interest by any statistical assay known in the art, such as A statistically significant number of patients as measured by, for example, Student's t-test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Johnkier-Tufstra test, and Wilcoxon test should reduce the symptoms of the disease of interest.

As used throughout the specification and claims, the term "consisting essentially of, or variations thereof, includes all described elements or groups of elements and does not significantly alter the basic or novel properties of a given administration regimen, method or composition. not, in fact, meant to optionally include other elements similar or different from those described. As a non-limiting example, a binding compound consisting essentially of a recited amino acid sequence may also comprise one or more amino acids that do not significantly affect the properties of the binding compound.

The term “natural” as applied to some objects of the present invention refers to the fact that such objects can be found in nature. For example, a polypeptide sequence or polynucleotide sequence is considered natural if it exists in an organism (including viruses), is isolated from a natural source, and has not been intentionally modified artificially in the laboratory.

An “effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. Said effective amount also refers to an amount sufficient to permit or facilitate diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition being treated, the patient's general health, the method, route and dosage, and severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

“Exogenous” refers to an organism, cell, or material produced outside the body according to the background. "Endogenous" refers to a cell, organism, or material produced inside the human body depending on the background.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. A molecule is considered homologous at that position if a position in two aligned sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by an adenine. . The percent homology between two sequences is a function of the number of coincident or homologous positions shared by the two sequences divided by the number of aligned positions multiplied by 100%. For example, in an optimal sequence alignment, if 6 out of 10 positions of the two sequences are congruent or homologous, then the two sequences are 60% homologous. In general, such alignments are made when the two sequences are aligned to obtain the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" may be used interchangeably, and all such designations include their progeny. Accordingly, the words "transformants" and "transformed cells" include primary test cells, and cultures cultured therefrom, regardless of the number of passages. It should also be noted that, due to intentional or unintentional mutation, not all progeny may be exactly identical in terms of DNA content. Mutant progeny that have the same function or biological activity as were selected for in the originally transformed cell are included. When different names are referred to, they may be clearly understood from the context.

"optionally" or "optionally" means that the event or circumstance following the word "optionally" or "optionally" may, but need not, occur, and that the description includes instances in which said event or circumstance occurs or does not occur . For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

A “pharmaceutical composition” refers to one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components as well as other components such as a physiologically/pharmaceutically acceptable carrier and mixtures containing excipients. The purpose of the pharmaceutical composition is to facilitate drug administration to an organism, promote absorption of an active ingredient and thereby exert biological activity. The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

"Pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present invention that is safe and effective for use in mammals and has the desired biological activity. The antibody-drug conjugates of the present invention contain one or more amino groups, and thus the conjugates are capable of forming salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate , dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate.

“Solvate” refers to a pharmaceutically acceptable solvate formed by an antibody-drug conjugate compound of the invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol and ethyl acetate.

"Cytotoxic drug", as used herein, refers to a substance that inhibits the function of a cell and/or causes cell death or destruction.

"Tubulin inhibitor" refers to a class of compounds that exert anti-tumor effects by inhibiting the polymerization of tubulin or promoting aggregation of tubulin, thereby interfering with the process of cell mitosis. Non-limiting examples thereof include maytansinoids, calcheamicin, taxane, vincristine, colchicine, dolastatin/auristatin/monomethyl auristatin E (MMAE)/monomethyl auristatin F (MMAF) do.

“Linker” refers to a chemical moiety that covalently attaches an antibody to a covalent bond or atomic chain of a drug. Non-limiting examples of linkers include arylene, heteroarylene, PEG, polymethyleneoxy, succinate, succinamide, diglycolate, malonate, and caproamide.

“Drug load” (DAR) is denoted by y, which is the average number of cytotoxic drugs per antibody in Formula A. In the present invention, the range of drug loading may be 1-20 cytotoxic drugs (D) per antibody. An antibody-drug conjugate of formula (A) is a collection of antibodies conjugated to a specific range (1-20) of a cytotoxic drug. The drug load (DAR) in the antibody-drug conjugate from the coupling reaction can be characterized by conventional means such as mass spectrometry, HPLC and ELISA. By this means, it is possible to determine the quantitative distribution of the antibody-drug conjugate with respect to the y value.

2. Abbreviations

MC = 6-maleimidohexanoyl

VC = valine-citrulline

PAB = p-aminobenzyloxycarbonyl

MMAE = monomethyl auristatin E (MW 718)

MMAF = variant of monomethyl auristatin E with phenylalanine at the C-terminus of the molecule (MW 731.5)

Examples are included below for further description of the invention, but these examples do not limit the scope of the invention. Experimental methods having conditions not specified in the examples of the present invention generally follow conventional conditions, for example, Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Follow the conditions recommended by the raw material or raw material manufacturer. Reagents with unspecified sources are commercially available conventional reagents.

Example 1: Antigen Preparation and Stable Cell Line Construction

The sequence encoding his-tagged human B7-H4 (huB7-H4-His) and the sequence encoding Fc-tagged human B7-H4 (huB7-H4-Fc) were obtained from CRO's Integrated DNA Technology (IDT) (Integrated DNA Technology (IDT)) and (the template sequence of the B7-H4 recombinant protein was all designed by the present invention) and each cloned into a pTT5 vector (Biovector). After the recombinant B7-H4 protein was expressed in 293T cells, it was purified by the following experimental method, and the purified protein could be used in the following experiments of Examples.

The sequence of huB7-H4-His:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKADYKDDDDKGSHHHHHHHH SEQ ID NO: 1

Sequence of huB7-H4-Fc:

FGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKAGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2

Purification steps of huB7-H4-His:

HEK293 cell expression supernatant samples were centrifuged at high speed to remove impurities. The buffer was replaced against PBS and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with a PBS solution containing 5 mM imidazole and washed with 2-5 times the column volume. A sample of the supernatant obtained after replacement was loaded onto the column. The column was washed with a PBS solution containing 5 mM imidazole until the A280 reading fell to baseline. The chromatography column was then washed with PBS+10 mM imidazole to remove non-specifically bound protein impurities, and the eluate was collected. The target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peak was collected. The collected eluate was further purified by ion exchange (SP column). Preparation of solution A: 0.01 M PB, pH 8.0. Prepare solution B: solution A + 1 M NaCl. First, the PBS solution of imidazole with the eluted target protein was replaced for solution A, the SP column was equilibrated using solution A and the sample was loaded. The concentration gradient of solution B was 0-100%. Samples were eluted at 10 times the column volume and each elution peak was collected. The obtained protein was confirmed as correct by electrophoresis, peptide mapping and liquid chromatography-mass spectrometry (LC-MS), and then aliquoted for use.

Purification steps of huB7-H4-Fc:

HEK293 cell expression supernatant samples were centrifuged at high speed to remove impurities. The buffer was replaced against PBS. The Protein A affinity column was equilibrated with 10 mM PBS buffer and washed with 2-5 times the column volume. A sample of the supernatant obtained after replacement was loaded onto the column. The column was washed with 25 times the column volume of buffer until the A280 reading fell to baseline. The target protein was eluted with 0.8% acetate buffer at pH 3.5 and the elution peak was collected. After aliquots, 1 M Tris-Cl pH 8.0 buffer was added immediately for neutralization. The solution was then exchanged against PBS pH 6.9 using an Amico-15 filter column from Millipore. The obtained protein was confirmed by electrophoresis, peptide mapping and liquid chromatography-mass spectrometry (LC-MS), and then aliquoted for use.

Composition of the stable CHO-S cell pool:

The full-length sequence encoding the human or cynomolgus monkey B7-H4 protein (huB7-H4 or cyB7-H4) was synthesized by Integrated DNA Technology (IDT) (the template sequence of the B7-H4 recombinant protein is shown in all Each was cloned into the modified pcDNA3.1 vector, pcDNA3.1/Puro (Invitrogen #V79020). CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, #10743029) at 0.5x10 ⁶ cells/ml. 10 μg of vector encoding the huB7-H4 or cyB7-H4 gene was mixed with 50 μl LF-LTX (Life Technologies, #A12621) in 1 ml of Opti-MEM medium (Life Technologies, #31985088). The mixture was incubated at room temperature for 20 minutes and added to the culture medium of CHO cells. Cells were placed in a carbon dioxide incubator for culture. After 24 hours, the culture medium was replaced with fresh medium and 10 μg/ml puromycin was added. Thereafter, the culture medium was replaced with fresh medium every 2-3 days, and a stable pool of CHO-S cells was obtained after 10-12 days of selection.

Example 2: Acquisition of mouse hybridomas and antibody sequences

A total of 5 Balb/c and 5 A/J female 10-week-old mice were immunized with the human antigen huB7-H4-Fc. Sigma complete Freund's adjuvant (CFA) and Sigma incomplete Freund's adjuvant (IFA) were used. A stable "water-in-oil" liquid was prepared by thoroughly mixing the immunogen and adjuvant and emulsifying in a 1:1 ratio. The injection dose was 25 μg/200 μl/mouse.

Day 01: 1st immunization, complete Freund's adjuvant.

Day 21: 2nd immunization, incomplete Freund's adjuvant.

Day 35: 3rd immunization, incomplete Freund's adjuvant.

Day 42: Blood sampling and serum titer testing (blood after 3 immunizations).

Day 49: 4th immunization, incomplete Freund's adjuvant.

Day 56: Blood sampling and serum titer testing (blood after 4 immunizations).

Affinity of antibody or serum was detected using an indirect ELISA method: huB7-H4-His protein was diluted with PBS pH 7.4 to a concentration of 1 μg/ml, and 96-well high-affinity ELISA at 100 μl/well. Plates were applied and stored refrigerated at 4°C for overnight (16-20 hours) incubation. Plates were washed 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20). A 3% bovine serum albumin (BSA) blocking solution diluted with PBST was added at 150 μl/well and incubated for 1 hour at room temperature for blocking. After blocking was complete, the blocking solution was discarded and the plate washed 4 times with PBST buffer. The antibody or serum to be tested is diluted with PBST containing 3% BSA to obtain a gradient of 10 doses (10-fold dilution) starting from 1 μM, which is added to microtiter plates at 100 μl/well and 1 at room temperature. incubated for hours. After completion of the incubation, the plates were washed 4 times with PBST and 100 μΐ// It was added to the wells and incubated for 1 hour at room temperature. Plates were washed 4 times with PBST, then TMB chromogenic substrate (Cell Signaling Technology, cat#7004S) was added at 100 μl/well and incubated for 1 minute in the dark at room temperature. Stop solution (Cell Signaling Technology, cat#7002S) was added at 100 μl/well to terminate the reaction. Absorbance values were read at 450 nm with a microplate reader (BioTek, Synergy H1 model). The data were analyzed to obtain the binding affinity of the antibody or serum to the human B7-H4 antigen.

Serum titers and the ability of immunized mouse sera to bind cell surface antigens were assessed using an indirect ELISA method. Cell fusion was performed according to the detection result of titer (>100,000 fold dilution). Immunized mice with high serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed. Splenocytes and SP2/0 myeloma cells were fused and plated to obtain hybridomas. Target hybridomas were selected by indirect ELISA and monoclonal cell lines were established by limit-dilution methods. Among the obtained positive antibody strains, hybridoma strains expressing non-specific binding antibodies were compared with CHO-S cells stably expressing huB7-H4 and cyB7-H4 with blank CHO-S cells. further excluded. Target hybridomas were selected using a method similar to that of the in vitro cell binding experiment of Example 5, and established as a monoclonal cell strain by the limit-dilution method. Hybridoma cells in the logarithmic growth phase were collected. RNA was extracted with Trizol (Invitrogen, 15596-018) and subjected to reverse transcription (PrimeScript® Reverse Transcriptase, Takara #2680A). The dDNA obtained by reverse transcription was amplified by PCR with a mouse Ig-primer set (Novagen, TB326 Rev.B 0503) and sequenced. Finally, the sequences of four strains of murine antibodies were obtained for humanization and construction of antibody-drug conjugates.

The heavy and light chain variable region sequences of the murine monoclonal antibody 2G6 are as follows:

2G6 HCVR

EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPEKRLEWVAGINGGGSYTYYLDTVKGRFTISRDNSRNTLYLQMSSLRSEDTAMYYCVSQGSNYYFDYWGQGTTLTVSS SEQ ID NO:46

2G6 LCVR

DIRMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKALIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK SEQ ID NO:47.

The heavy and light chain variable regions of murine monoclonal antibody 2G6 comprise the following CDR sequences:

The sequences of the heavy and light chain variable regions of the murine monoclonal antibody 2F7 are as follows:

2F7 HCVR

EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLKPEDTAMYYCTRESYSQGNYFDYWGQGTTLTVSS SEQ ID NO: 48

2F7 LCVR

DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFSLTFGAGTKLELK SEQ ID NO: 49.

The heavy and light chain variable regions of murine monoclonal antibody 2F7 comprise the following CDR sequences:

The sequences of the heavy and light chain variable regions of the murine monoclonal antibody 2F8 are as follows:

2F8 HCVR

QVQLQQPGSVLVRPGASVKLSCKASGYTFTNSWMNWAKLRPGQGLEWIGGIYPNSGNIEYNEKFKGKATLTVDTSSSTAYMDLTSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO: 50

2F8 LCVR

DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQSPKLLISSTSYRYTGVPDRFTGSGSGTDFTFIISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK SEQ ID NO: 51.

The heavy and light chain variable regions of murine monoclonal antibody 2F8 comprise the following CDR sequences:

The sequences of the heavy and light chain variable regions of the murine monoclonal antibody 1C9 are as follows:

1C9 HCVR

QVQLQQPGSVLVRPGASVKLSCKASGDTFTTYWMNWVKQRPGQGLEWIGGIYLNSGSSEYNEKFKGKATLSVDTSSSTAYMDLSSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO: 52

1C9 LCVR

DIVMTQSHKFLSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPELLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYNTPLTFGAGTQLELK SEQ ID NO:53.

The heavy and light chain variable regions of murine monoclonal antibody 1C9 comprise the following CDR sequences:

Example 3: Humanization experiment of murine antibody

Humanization of the murine anti-human B7-H4 monoclonal antibody was performed using methods as published in numerous documents in the art. Briefly, the parent (murine antibody) constant domain was replaced with a human constant domain. In the present invention, human germline antibody sequences were selected according to the homology between murine and human antibodies, and murine antibodies 2G6, 2F7, 2F8 and 1C9 were humanized. The CDR regions of murine antibodies 2G6 and 2F7 were grafted onto the selected corresponding humanized templates to replace the humanized variable regions, followed by recombination with an IgG constant region (preferably IgGl for heavy chain, and κ for light chain). made it Then, based on the three-dimensional structure of the murine antibody, built-in residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of VL and VH were reverse mutated, and chemically unstable amino acid residues of the CDR regions were Optimized, wherein HCDR1 of murine monoclonal antibody 2F8 was optimized for GYTFTSSWMN (SEQ ID NO: 43), HCDR2 was optimized for GIYPNRGNIEY NEKFKG (SEQ ID NO: 44), and HCDR2 of murine monoclonal antibody 1C9 was optimized for YLNRGS (SEQ ID NO: 45) , A designed humanized antibody comprising a combination of the following light and heavy chain variable region sequences was obtained.

The heavy and light chain variable regions of the humanized antibody hu2G6 are as follows:

hu2G6 HCVR

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSS SEQ ID NO: 15

hu2G6 LCVR

DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIK SEQ ID NO: 16

The heavy and light chain variable regions of the humanized antibody hu2F7 are as follows:

hu2F7 HCVR

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSS SEQ ID NO: 17

hu2F7 LCVR

EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK SEQ ID NO: 18.

The heavy and light chain variable regions of the humanized antibody hu2F8 are as follows:

hu2F8 HCVR

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO: 35

hu2F8 LCVR

DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK SEQ ID NO: 36

The heavy and light chain variable regions of the humanized antibody hu1C9 are as follows:

hu1C9 HCVR

EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO:37

hu1C9 LCVR

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIK SEQ ID NO: 38.

The variable regions were recombined with an IgG constant region (preferably IgGl for heavy chain, and κ for light chain). Exemplary heavy and light chain constant region sequences are shown below:

IgG1 C:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 54

Ig Kappa C:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 55.

A humanized antibody consisting of the following exemplary light and heavy chains was obtained after ligation:

hu2G6 HC

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 19

hu2G6 LC

DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTYVCLLNNFYPREAKVQNSFTLNNFYPREAKVQNSFTLNNFYPREAKVQSSQPEDFATYYCVQYAQFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTYVCLLNNFYPREAKVQNSFTLNNFYPREAKVQNSQVKVDNALQSSKHSG

SEQ ID NO: 20

hu2F7 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 21

hu2F7 LC

EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLSSECTLSYPREAKVQWKVACEKATEKLQSGNSQESNRFYPREAKVQWKVEKVEKLQSGNSQESVNRTEGLQSSKPSTY

SEQ ID NO: 22

hu2F8 HC

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 39

hu2F8 LC

DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTKVCLLNNFYPREAKVQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTKVCLLNNFYPREAKVQQKPGKAPKLLISSTSKVQNLNNFYPREAKVQWYESVTHQQDSSGKV

SEQ ID NO: 40

hu1C9HC

EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 41

hu1C9 LC

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTKVCLLNNFYPREAKVQQKPGKAPKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVTYVCLLNNFYPREAKVQQKPGKAPKALQSSKNSQNRSVTHQDALQSSKNSQ

SEQ ID NO: 42

Example 4: Expression of anti-B7-H4 humanized antibody

Recombinant vectors were designed according to the sequences of humanized antibodies. The heavy chain vector was designed as follows: signal peptide + humanized heavy chain. The light chain vector was designed as follows: signal peptide + humanized light chain.

This sequence was inserted into the pCEP4 vector (ThermoFisher #V04450). Expression vectors were synthesized according to the design above. Vector plasmids were obtained, extracted on a large scale and sent for sequencing for validation. Competent plasmids were transfected into human 293F cells (Thermo Fisher # R79007) with PEI (Thermo Fisher # BMS1003-A). 293F cells were continuously cultured in logarithmic growth phase with serum-free medium (Shanghai OPM Biosciences, OPM-293 CD03) and used for cell transfection. 21.4 μg of humanized antibody light chain plasmid and 23.6 μg of humanized antibody heavy chain plasmid were dissolved in 10 ml Opti-MEM® I reduced serum medium (GIBCO, 31985-070), mixed well, and then 200 μg of PEI was added, mixed well, and incubated for 15 minutes at RT. Then 50 ml of cells were added. Cell culture conditions: 5% CO ₂ , 37° C., 125 rpm/min. During culture, supplements were added on days 1 and 3 until cell viability was less than 70%. Cell supernatant was collected, centrifuged and filtered. The centrifuged and filtered cell culture medium was loaded onto an antibody purification affinity column. The column was washed with phosphate buffer. Samples were eluted with glycine hydrochloride buffer (pH 2.7, 0.1 M Gly-HCl), neutralized with 1 M Tris hydrochloride pH 9.0, and dialyzed against phosphate buffer to give the final purified humanized antibody, which in each run It could be used for ceremonial experiments.

Example 5: Detection of In Vitro Binding Capacity of Humanized Antibodies

The designed humanized antibody was tested in an in vitro experiment as follows:

1. In vitro cell binding experiments:

Cultured MX-1 cells expressing human B7-H4 (CLS Cell Lines Service GmbH #300296) were harvested, the cell density was adjusted to pH 7.4 with PBS, and the cells were washed with V-shaped bottoms. In 96-well plates with 1x10 ⁵ cells per well. The plate was centrifuged at 2000 rpm for 5 minutes and the supernatant removed. 100 μl of gradient diluted humanized antibody solution (diluted with PBS containing 0.5% BSA, starting at 1 μM, 3-fold gradient of 10 doses) is added to each well, mixed well and at 4° C. on a shaker. Incubated for 1 hour. The plate was centrifuged at 2000 rpm for 5 minutes and the supernatant removed. Cells were washed twice with PBS. FITC-labeled goat anti-human secondary antibody (Abcam, cat#ab97224) diluted in 100 μl of 0.5% BSA in PBS was added to each well, mixed well and incubated for 30 minutes at 4° C. on a shaker. . The plate was centrifuged at 2000 rpm for 5 minutes and the supernatant was removed. Cells were washed twice with PBS and then resuspended in PBS. Signals were detected using a flow cytometer (BECKMAN COULTER, model DxFLEX) and concentration curves were plotted for analysis of the results. The results are as shown in Table 1. Both humanized antibodies hu2G6 and hu2F7 positively bind to MX-1 cells with high expression of B7-H4.

Affinity of each humanized antibody for MX-1 cells (EC ₅₀ ) Antibody name FACS binding EC ₅₀ (nM) with MX-1 cells hu2G6 14.3 hu2F8 7.26 hu1C9 7.25 hu2F7 7.32

2. Affinity Kinetics Experiment:

The Biacore method is a generally accepted method for objectively detecting affinity and kinetics between proteins. The affinity and binding kinetics of the antibodies to be tested of the present invention were analyzed by Biacore T200 (GE). The anti-B7-H4 antibody to be tested of the present invention was covalently linked to a CM5(GE) chip using the kit provided by Biacore and the NHS standard amino coupling method. 50 nM of human huB7-H4-His protein diluted in the same buffer was then injected at a flow rate of 10 uL/min. After injection, all samples were regenerated with the regeneration reagent in the kit. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. The acquired data were analyzed with a 1:1 (Langmuir) binding model using GE's BIAevaluation software. The affinity kinetic data of the humanized antibody calculated by the above method are shown in Table 2. The humanized antibodies hu2G6, hu2F7, hu2F8 and hu1C9 all show strong affinity for the human B7-H4 antigen protein.

Affinity and kinetic characterization of each humanized antibody antibody bonding speed
k _a (1/M*s) dissociation rate
k _d (1/s) affinity
K _D hu2G6 7.41e+05 1.00e-05 1.35e-11 hu2F8 4.47e+05 1.12e-04 2.50e-10 hu1C9 3.35e+05 1.00e-05 2.98e-11 hu2F7 3.29e+05 2.49e-04 7.57e-10

Example 6: Endocytosis of anti-B7-H4 antibody

To test whether the antibody of the present invention can be endocytosed together with human B7-H4 after binding to B7-H4, MX-1 cells were used for evaluation. MX-1 cells were trypsinized (first washed once with PBS at 37° C. for about 2 minutes), collected and resuspended in pre-chilled FACS buffer. The cell concentration was adjusted to 1x10 ⁶ cells/ml. 1 ml of the cell suspension was added to the EP tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Cells were resuspended by adding 1 ml of the prepared antibody to be tested, and the final concentration of the antibody was 20 μg/ml. Cells were incubated for 1 h on a 4°C shaker, centrifuged, and the supernatant discarded (4°C, 1500 rpm×5 min). Cells were washed twice with FACS buffer and the supernatant removed. Cells were resuspended by adding 100 μl of fluorescent secondary antibody working solution to each tube. Cells were incubated for 30 min on a 4°C shaker, centrifuged and the supernatant discarded (4°C, 1500 rpm×5 min). Cells were washed twice with FACS buffer and the supernatant removed. 1.0 ml of prewarmed MX-1 cell complete medium was added to each tube to resuspend and mix the cells. The cell suspension was aliquoted into 4 tubes at 200 μl per tube (these were the 0 min group, blank group, 30 min group and 2 h group, respectively). The 0 min and blank groups were placed on ice, while the other groups were placed in a 37° C. incubator for endocytosis for 30 min and 2 h, respectively. At the corresponding time point, the EP tube was removed and placed on ice to pre-cool for 5 minutes. All treatment groups were centrifuged and the supernatant discarded (4° C., 1500 rpm×5 min). Cells were washed once with FACS buffer and the supernatant removed. 250 μl strip buffer was added to the EP tubes of all treatment groups except for the 0 min group and incubated at room temperature for 8 minutes. The cells were centrifuged and the supernatant discarded (4° C., 1500 rpm×5 min). Cells were washed twice with FACS buffer and the supernatant removed. 100 μl immunostaining fixative was added to all treatment groups, placed at 4° C. for over 30 minutes, and detected by flow cytometry DxFlex. B7-H4 antibody endocytosis percentage (%) = (mean fluorescence intensity value at each time point - mean fluorescence intensity value of blank group) / (mean fluorescence intensity value at time point 0 - mean fluorescence intensity value of blank group) x 100%. Data are shown in Table 3.

Humanized Antibody-Mediated Endocytosis Efficiency of B7-H4 Proteins antibody 0 hours (%) 0.5 hours (%) 2 hours(%) IgG control 0 0.1 0.9 hu2G6 0 18.6 28.7 hu2F7 0 13.9 25.9

Example 7: Conjugation of Antibodies to MC-MMAF

The antibodies of the present invention have cell affinity activity and endocytosis activity, which makes the antibody suitable for coupling with drugs to form antibody-drug conjugates for the treatment of B7-H4-mediated diseases. The coupling process is shown in the following formula, wherein Ab represents hu2G6 or hu2F7:

In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 ml acetonitrile to form a solution for later use. The above-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate was added to the antibody in pH=4.3 acetate/sodium acetate buffer (10.35 mg/ml, 9.0 ml, 0.97 mol). Then, 1.0 ml of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise to the reaction mixture and reacted at 25° C. for 2 hours under shaking. After completion of the reaction, the reaction mixture was desalted and purified using Sephadex G25 gel column (eluted phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 1f. The solution was concentrated to 10 mg/ml and used directly in the next reaction.

In the second step, 1f solution (11.0 ml) was added together with 0.35 ml of 2.0 M carboxyamine hydrochloride solution and reacted at 25° C. for 30 minutes under shaking. Then, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluted phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 2f (concentration 6.17 mg/ml, 14.7 ml).

In the third step, the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 ml acetonitrile and in solution 2f (concentration 6.17 mg/ml, 3.0 ml) and reacted at 25° C. for 4 hours under shaking. The reaction solution was then desalted and purified by Sephadex G25 gel column (eluent phase: pH 6.5 0.05 M PBS solution), filtered under sterile conditions and the product Ab-MC-MMAF antibody-drug conjugate (3.7 mg) in PBS buffer. /mL, 4.7 mL) was obtained, which was refrigerated at 4°C. The average value y of the product hu2G6-MC-MMAF measured by HIC-HPLC was 3.8, and a sample of hu2G6-MC-MMAF (y=4) was obtained by HIC-HPLC purification. The average value y of the product hu2F7-MC-MMAF measured by HIC-HPLC was 3.2, and samples of hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) were analyzed by HIC-HPLC purification. obtained.

Example 8: Conjugation of Antibody to SN-38

Antibody-conjugated drugs were prepared via the following coupling procedure, where Ab represents hu2F7:

In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 ml acetonitrile to form a solution for later use. The above-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate was added to the antibody in pH=4.3 acetate/sodium acetate buffer (10.35 mg/ml, 9.0 ml, 0.97 mol). Then, 1.0 ml of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise to the reaction mixture and reacted at 25° C. for 2 hours under shaking. After completion of the reaction, the reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 1h. The solution was concentrated to 10 mg/ml and used directly in the next reaction.

In the second step, 1 h solution (11.0 ml) was added together with 0.35 ml of 2.0 M carboxyamine hydrochloride solution and reacted at 25° C. for 30 minutes under shaking. Then, the reaction solution was desalted and purified using a Sephadex G25 gel column (eluted phase: pH 6.5 0.05 M PBS solution) to obtain a solution of the product 2h (concentration 6.2 mg/ml, 15.0 ml). The 2h solution was concentrated to about 10 mg/ml and used in the next reaction.

In the third step, the compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 mol) was dissolved in 0.3 ml acetonitrile, added to a 2h solution (concentration 6.2 mg/ml, 3.0 ml) and 4 at 25°C. The reaction was carried out under shaking for hours. The reaction solution was then desalted and purified by Sephadex G25 gel column (eluent phase: pH 6.5 0.05 M PBS solution), filtered under sterile conditions and the product hu2F7-SN-38 antibody-drug conjugate (3.7 mg) in PBS buffer. /mL, 4.7 mL) was obtained, which was refrigerated at 4°C. The average value y was measured by the ultraviolet method. The cuvette filled with sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after deducting the solvent blank, the cuvette filled with the test solution was placed in the sample measurement absorption cell. Absorbance at 280 nm and 370 nm was measured.

Data processing:

The antibody content C _mab was determined by establishing a standard curve and measuring the absorbance at a wavelength of 280 nm. The small molecule content C _Drug was determined by measuring the absorbance at a wavelength of 370 nm.

Mean drug load y = C _Drug /C _mab

The average value y = 3.7 of the product hu2F7-SN-38 was determined by this method. A sample of hu2F7-SN-38 (y=4) was obtained by UV-HPLC purification.

Example 9: Conjugation of Antibodies to Exatecan

In step 1, 2a (2 g, 17.2 mmol) was dissolved in 75 mL acetonitrile and potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol) and tetrabutylammonium iodide (620 mg) , 1.68 mmol) were added successively. The reaction solution was stirred at room temperature for 48 h and filtered through diatomaceous earth. The filter cake was washed with ethyl acetate (20 mL). The filtrates were combined and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with a developing solvent system C to give the product 5a (3.2 g, yield: 90.1%).

In step 2, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to the reaction flask, 6 ml tetrahydrofuran was added and replaced with argon three times. The reaction mixture was cooled to 0-5° C. in an ice-water bath and potassium tert-butoxide (164 mg, 1.46 mmol) was added, then the ice bath was removed, allowed to warm to room temperature and stirred for 40 minutes. To the reaction mixture, 15 ml of ice water was added, followed by extraction with ethyl acetate (40 ml x 2) and chloroform (20 ml x 5). The organic phases were combined and concentrated. The obtained residue was dissolved in 6 mL dioxane, 3 mL water, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorene methyl chloroformate (190 mg, 0.734 mmol) were added, followed by stirring at room temperature for 2 hours. . 30 ml of water was added to the reaction solution, followed by extraction with ethyl acetate (20 ml x 3). The organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with a developing solvent system C to produce product 5b benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9 -Dioxa-4,7-diazaundec-11-ate (73 mg, yield: 19.4%) was obtained.

MS m/z (ESI): 515.0 [M+1].

In step 3, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 ml of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2: 1), palladium on carbon (18 mg, content 10%, dry basis), replaced with hydrogen 3 times, and reacted with stirring at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate. Concentrate the filtrate to obtain crude product 5c, 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11- The acid (20 mg) was obtained, which was used directly in the next reaction without purification.

MS m/z (ESI): 424.9 [M+1].

In step 4, 1b (15 mg, 28.2 μmol) was added to the reaction flask, 1.5 ml N,N-dimethylformamide was added and replaced with argon 3 times. The reaction mixture was cooled to 0-5° C. in an ice-water bath, 1 drop of triethylamine was added, crude product 5c (20 mg, 47.1 μmol) was added, and 4-(4,6-dimethoxy-1,3 ,5-triazin-2-yl)-4-methylchloromorpholine (25.4 mg, 86.2 μmol) was added, and the reaction was stirred in an ice bath for 40 minutes. 15 ml of water was added to the reaction mixture, followed by extraction with ethyl acetate (20 ml x 3). The organic phase was collected. The organic phase was washed with saturated sodium chloride solution (20 mL x 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The obtained residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 5d (9H-fluoren-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S) )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo [de] pyrano[3',4':6,7]indologino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (23.7 mg, yield: 78.9%) was obtained.

MS m/z (ESI): 842.1 [M+1].

In step 5, 5d (30 mg, 35.7 μmol) was dissolved in 3 ml dichloromethane, 1.5 ml diethylamine was added and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1.5 ml toluene was added, and concentrated under reduced pressure, which was repeated twice. 4.5 ml n-hexane was added to the residue and it was pulped. After standing, the supernatant was poured and the solid was maintained. The solid residue was concentrated under reduced pressure and pumped to dryness to give crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6 ,7]indologino[1,2-b]quinolin-1-yl)acetamide (23 mg) was obtained, which was used directly in the next reaction without purification.

MS m/z (ESI): 638.0 [M+18].

In step 6, the crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 ml N,N-dimethylformamide and replaced with argon three times. The reaction mixture was cooled to 0-5° C. in an ice-water bath, 0.5 ml of 4 g (31.8 mg, 67.3 μmol) of N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy- 1,3,5-triazin-2-yl)-4-methylchloromorpholine (27.8 mg, 94.3 μmol) was added and reacted with stirring in an ice bath for 10 minutes. The reaction mixture was warmed to room temperature by removing the ice bath and reacted under stirring for 1 hour to give compound 5. The reaction solution was subjected to high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc): B-acetonitrile, gradient elution, flow rate: 18 mL /min). The corresponding components were collected and concentrated under reduced pressure to give products 5-A and 5-B (3.6 mg, 2.6 mg).

MS m/z (ESI): 1074.4 [M+1].

Single configuration compound 5-A (shorter retention time)

UPLC analysis: retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02 7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65 5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 2H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 2H), 2.80-2.62 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.15 (m) , 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H).

Single configuration compound 5-B (longer retention time):

UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm; mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H) ), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H) , 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

For the preparation of other intermediates, refer to the method of intermediate 5.

Tris(2-carboxyethyl)phosphine (10 mM, 0.347 ml, 3.47) in PBS buffered aqueous solution of antibody hu2F7 (pH = 6.5 0.05 M PBS buffered aqueous solution; 7.3 ml, 13.8 mg/ml, 0.681 μmol) at 37°C μmol) of the prepared aqueous solution was added. The reaction mixture was placed in a water bath shaker and reacted at 37° C. under shaking for 3 hours. After the reaction was terminated, the reaction solution was cooled to 25° C. in a water bath, diluted to 14.0 ml, and 3.3 ml of the solution was taken for the next reaction.

Compound 5-A (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL DMSO and added to the 3.3 mL solution. The reaction mixture was placed on a water bath shaker and stirred at 25° C. for 3 hours under shaking. The reaction was terminated. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent phase: pH 6.5 0.05 M PBS buffered aqueous solution, containing 0.001 M EDTA) to Ab-exatecan in PBS buffer (1.35 mg/ml, 13 ml). , obtained an exemplary product of hu2F7-exatecan (Compound 34), which was stored at 4°C. The average value y was measured by the ultraviolet method. The cuvette filled with sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after deducting the solvent blank, the cuvette filled with the test solution was placed in the sample measurement absorption cell. Absorbance at 280 nm and 370 nm was measured.

Data processing:

The antibody content C _mab was determined by establishing a standard curve and measuring the absorbance at a wavelength of 280 nm. The small molecule content C _Drug was determined by measuring the absorbance at a wavelength of 370 nm.

Mean drug load y = C _Drug /C _mab

For the exemplary product hu2F7-exatecan (Compound 34), y was determined to be 7.6 by this method. A sample of hu2F7-exatecan (y=8) was obtained by UV-HPLC purification.

For the preparation of other antibody conjugates, refer to the method of compound 34.

Example 10: Killing activity of antibody-drug conjugates against tumor cells

To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, MX-1 breast cancer cells were used for evaluation. MX-1 cells were harvested, centrifuged and counted. Cell density was adjusted to 0.44× ^{10 6} cells/ml with complete medium, and cells were plated in 60 wells of white 96-well plates at 90 μl per well at a cell count of 40,000. 100 μl PBS was added to the remaining peripheral wells. The cell plate was placed in an incubator at 37° C., 5% CO ₂ and incubated overnight. On day 2 of the experiment, antibody-drug conjugate solutions were prepared in PBS in 96-well V-bottom plates, starting at a concentration of 1000 nM (3-fold dilution and 9 concentrations). After completion of the preparation, the solution was added to white 96-well plates in duplicate at 10 μl per well. The cell plate was placed in a 37° C., 5% CO ₂ incubator and the culture was continued for 72 hours. On day 5 of the experiment, plates were detected and read. The cell culture plate was removed. Two control wells were set up. 100 μl medium containing 40,000 cells was added to each well, and after equilibration to room temperature, 50 μl CTG solution (Promega G7573) was added to each well. The mixture was shaken and mixed, placed in the dark and left for 10 minutes, then detected using the microplate reader's luminescence program. Maximum killing rate = (fluorescence value of 1-1000 nM wells/fluorescence value of control well)%. The experimental results are shown in Table 4:

Evaluation of the killing activity of antibody-drug conjugates on tumor cells Antibody-Drug Conjugates IC ₅₀ (nM) Killing effect at maximum dose IgG-MC-MMAF (control) ND ND hu2G6-MC-MMAF (compound 1, y=4) 1.1 48.2% hu2F7-MC-MMAF (compound 2, y=4) 3.4 53.9% hu2F7-SN-38 (compound 6, y=4) 78.85 56.4% hu2F7-Exatecan (Compound 34, y=8) 80.28 60.39% ND: no detectable activity

Example 11: Inhibitory effect of antibody-drug conjugates on the growth of tumor cells

In order to test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, SK-BR-3 (ATCC #HTB30) breast cancer cells were used for evaluation. SK-BR-3 cells were harvested, centrifuged and counted. Cell density was adjusted to 0.44× ^{10 6} cells/ml with complete medium, and cells were plated in 60 wells of white 96-well plates at 90 μl per well at a cell count of 40,000. 100 μl PBS was added to the remaining peripheral wells. The cell plate was placed in an incubator at 37° C., 5% CO ₂ and incubated overnight. On day 2 of the experiment, antibody-drug conjugate solutions were prepared in PBS in 96-well V-bottom plates, starting at a concentration of 1000 nM (3-fold dilution and 9 concentrations). After completion of the preparation, the solution was added to white 96-well plates in duplicate at 10 μl per well. Two other control wells were set up and 10 μl PBS was added to each well. The cell plate was placed in a 37° C., 5% CO ₂ incubator and the culture was continued for 72 hours. On day 5 of the experiment, plates were detected and read. The cell culture plate was removed. After equilibration to room temperature, 50 μl CTG solution (Promega G7573) was added to each well. The mixture was shaken and mixed, placed in the dark and left for 10 minutes, then detected using the microplate reader's luminescence program. Maximum inhibition = (fluorescence value of 1-1000 nM well/fluorescence value of control well)%. The experimental results are shown in Table 5:

Evaluation of inhibitory activity of antibody-drug conjugates against tumor cells Antibody-Drug Conjugates IC ₅₀ (nM) maximum inhibition rate IgG-MC-MMAF (control) ND ND hu2G6-MC-MMAF (compound 1, y=4) 1.0 90.3% hu2F7-MC-MMAF (compound 2, y=4) 9.5 91.8% ND: no detectable activity

Example 12: Evaluation of In Vivo Efficacy of Antibody-Drug Conjugates Conjugated to MMAF

After forming tumors transplanted with MX-1 cells in mice, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated. 5x10 ⁶ MX-1 cells were subcutaneously injected into immunodeficient nude mice (BALB/c Nude). After 2 weeks, intravenous infusions of the antibody-drug conjugates hu2G6-MC-MMAF and hu2F7-MC-MMAF were performed at a frequency of once per week and at a dose of 1.5 mg/kg or 3 mg/kg. Human IgG1 protein was used as a control at a dose of 3 mg/kg. There were 5 mice in each group of the control or administration group. Tumor inhibition was calculated by measuring tumor volume. Tumor inhibition = 100% - (tumor volume in dosing group on day 21 - tumor volume in dosing group on day 0)/(tumor volume in control group on day 21 - tumor volume in control group on day 0). Experimental results are as shown in FIG. 1 and Table 6. Both hu2G6-MC-MMAF and hu2F7-MC-MMAF show dose-dependent anti-tumor effects. Both hu2G6-MC-MMAF and hu2F7-MC-MMAF showed a tumor inhibition rate of more than 100% at a dose of 3 mg/kg, indicating that the antibody-drug conjugate of the present invention not only inhibits tumor growth, but also inhibits tumor growth in already formed tumors. This means that it can exert a killing effect on

Efficacy of Dosing Compounds on MX-1 Implanted Tumors in Tumor-bearing Nude Mice administration group tumor suppression rate hu2G6-MC-MMAF (compound 1, y=4) 1.5 mg/kg 38.0% hu2G6-MC-MMAF (compound 1, y=4) 3 mg/kg 115.6% hu2F7-MC-MMAF (compound 2, y=4) 1.5 mg/kg 90.3% hu2F7-MC-MMAF (compound 2, y=4) 3 mg/kg 163.8%

Example 13: Evaluation of In Vivo Efficacy of Antibody-Drug Conjugates with Different Drug Loads

To further study antibody-drug conjugates with different drug loadings, antibody-drug conjugates were prepared by the method of Example 7 and purified by HPLC to hu2F7-MC-MMAF (y=2) and hu2F7-MC- MMAF (y=4) was obtained ( FIG. 2 ). After forming tumors transplanted with MX-1 in mice, the anti-tumor effects of the antibody-drug conjugates of the present invention were evaluated. 5x10 ⁶ MX-1 cells were subcutaneously injected into immunodeficient nude mice. After 2 weeks, intravenous infusions of the antibody-drug conjugates hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) were performed at a frequency of once per week and a dose of 3 mg/kg. Human IgG1 protein was used as a control at a dose of 3 mg/kg. There were 5 mice in each group of the control or administration group. Tumor inhibition was calculated by measuring tumor volume. Tumor inhibition = 100% - (tumor volume in dosing group on day 21 - tumor volume in dosing group on day 0)/(tumor volume in control group on day 21 - tumor volume in control group on day 0). Experimental results are as shown in FIG. 3 and Table 7. Both hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) show anti-tumor effects. hu2F7-MC-MMAF (y=4) had a stronger anti-tumor effect than hu2F7-MC-MMAF (y=2) at a dose of 3 mg/kg, showing a tumor suppression rate of greater than 100%, which This means that hu2F7-MC-MMAF (y=4) can not only inhibit tumor growth, but also exert an apoptotic effect on already formed tumors (Fig. 4).

Efficacy of Antibody-Drug Conjugates with Different Drug Loads on MX-1 Implanted Tumors in Tumor-bearing Nude Mice administration group tumor suppression rate hu2F7-MC-MMAF (y=2) 3 mg/kg 87.99% hu2F7-MC-MMAF (y=4) 3 mg/kg 197.87%

Example 14: Evaluation of In Vivo Efficacy of Antibody-Drug Conjugates Conjugated to Exatecan

To study the antibody-drug conjugate conjugated to exatecan, the antibody-drug conjugate was prepared by the method of Example 9 and purified by HPLC to obtain hu2F7-exatecan (compound 34, y=8). . After forming tumors transplanted with MX-1 cells in mice, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated. 5x10 ⁶ MX-1 cells were subcutaneously injected into immunodeficient nude mice. After 2 weeks, intravenous infusion of the antibody-drug conjugate hu2F7-exatecan (y=8) was performed at a frequency of once per week and at a dose of 5 mg/kg or 10 mg/kg. Human IgG1 protein was used as a control at a dose of 5 mg/kg. There were 5 mice in each group of the control or administration group. Tumor inhibition was calculated by measuring tumor volume. Tumor inhibition = 100% - (tumor volume in dosing group on day 18 - tumor volume in dosing group on day 0)/(tumor volume in control group on day 18 - tumor volume in control group on day 0). The experimental results are as shown in Table 8. hu2F7-exatecan exhibits >100% tumor inhibition at both doses of 5 mg/kg and 10 mg/kg, indicating that hu2F7-exatecan (y=8) not only inhibits tumor growth, but also It means that it can exert a killing effect on the formed tumor.

Efficacy of antibody-drug conjugates conjugated to exatecan against MX-1 implanted tumors in tumor-bearing nude mice administration group tumor suppression rate hu2F7-exatecan (compound 34, y=8) 5 mg/kg 173.55% hu2F7-exatecan (compound 34, y=8) 10 mg/kg 183.48%

Example 15: Exatecan-conjugated antibody-drug conjugate experiment on bystander killing activity

To study the effect of an antibody-drug conjugate conjugated to exatecan of the present invention on B7-H4 negative cell killing via the bystander effect, killing of the antibody-drug conjugate against a myeloma cell line with negative B7-H4 expression Activity was detected using the ONE-Glo™ Luciferase Assay Detection Kit. MX-1 cells were detected, centrifuged, counted, adjusted to a cell suspension of 7.5x10 ⁶ cells/ml with complete medium, and plated onto white 96-well plates. 33 μl of the MX-1 cell suspension was added to each well of the “mixed cell group” and 33 μl PBS was added to each well of the “single cell group”. NCI-H929-LUC tumor cells with negative B7-H4 expression (Cobioer catalog number CBP30061L) were harvested. The cell density was adjusted to 1.5x10 ⁶ cells/ml. 33 μl of NCI-H929-LUC cell suspension was added to each well of “mixed cell group” and “single cell group”. The cell plate was placed in an incubator at 37° C., 5% CO ₂ and incubated overnight. On the second day of the experiment, the highest concentration of the antibody-drug conjugate hu2F7-exatecan (Compound 34, y=8) to be tested was uniformly adjusted to 3000 nM and diluted to obtain a 5-fold gradient of 9 doses. 33.3 μl of the diluted antibody-drug conjugate working solution was added to each well of the experimental plate and mixed gently. The cell plate was placed in an incubator at 37° C., 5% CO ₂ and incubated for 48 hours. The cell culture plate was removed and equilibrated to room temperature. Then, 100 μl of ONE-Glo (trademark) luciferase reagent (Promega E6120) was added to each well. Cells were shaken and lysed for 10 minutes in the dark at room temperature. Cell lysates were centrifuged at 1000 rpm/min for 1 min and then transferred to a clear bottom 96-well plate at 180 μl per well. The plate was read at a wavelength of 490 nm on a microplate reader. Data were analyzed using Graphpad Prism software, and the experimental results are shown in Table 9. ONE-Glo (trademark) luciferase reagent can specifically detect the viability of NCI-H929-LUC cells. hu2F7-exatecan effectively kills NCI-H929-LUC cells of "mixed cell population", which indicates that in the presence of B7-H4-positive cells, hu2F7-exatecan has an bystander killing effect, and B7-H4-positive This indicates that not only does it kill cells, but it can also kill insensitive B7-H4 negative cells (in the "single cell population").

Assessment of bystander killing activity experimental group IC ₅₀ (nM) maximum inhibition rate Mixed cell population (MX-1:NCI-LUC=5:1) 70.34 76.8% Single cell group (NCI-H929-LUC) ND ND ND: no detectable activity

SEQUENCE LISTING <110> SHANGHAI HANSOH BIOMEDICAL CO., LTD. JIANGSU HANSOH PHARMACEUTICAL GROUP CO., LTD. <120> ANTI-B7-H4 ANTIBODY-DRUG CONJUGATE AND MEDICINAL USE THEREOF <130> 702041CPCT <140> PCT/CN2020/094856 <141> 2020-06-08 <150> CN 201910498993.2 <151> 2019-06-06 < 150> CN 202010215322.3 <151> 2020-03-24 <160> 55 <170> SIPOSequenceListing 1.0 <210> 1 <211> 276 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huB7- H4-His <400> 1 Met Ala Ser Leu Gly Gin Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250 255 Lys Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser His His His His 260 265 270 His His His His 275 <210> 2 <211> 463 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huB7-H4-Fc <400> 2 Phe Gly Ile Ser Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser 1 5 10 15 Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro 20 25 30 Asp Ile Lys Leu Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val 35 40 45 Leu Gly Leu Val His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu 50 55 60 Gln Asp Glu Met Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val 65 70 75 80 Ile Val Gly Asn Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp 85 90 95 Ala Gly Thr Tyr Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn 100 105 110 Ala Asn Leu Glu Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn 115 120 125 Val Asp Tyr Asn Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg 130 135 140 Trp Phe Pro Gln Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly 145 150 155 160 Ala Asn Phe Ser Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu 165 170 175 Asn Val Thr Met Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn 180 185 190 Asn Thr Tyr Ser Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly 195 200 205 Asp Ile Lys Val Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln 210 215 220 Leu Leu Asn Ser Lys Ala Gly Ser Gly Gly Gly Gly Gly Asp Lys Thr His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 355 360 365 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN < 223> HCDR1 <400> 3 Gly Phe Thr Phe Ser Arg Tyr Gly Met Ser 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400 > 4 Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val Lys 1 5 10 15 Gly <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR3 <400> 5 Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 <400> 6 His Ala Ser Gln Gly Ile Ser Ser Asn Ile Gly 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR2 <400> 7 His Gly Thr Asn Leu Glu Asp 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> < 221> DOMAIN <223> LCDR3 <400> 8 Val Gln Tyr Ala Gln Phe Pro Tyr Thr 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR1 <400> 9 Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser 1 5 10 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400> 10 Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR3 <400 > 11 Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 <400> 12 Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR2 <400> 13 Phe Ala Ser Gln Ser Ile Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR3 <400> 14 Gln Asn Gly His Ser Phe S er Leu Thr 1 5 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu2G6 HCVR <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu2G6 LCVR <400> 16 Asp Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn 20 25 30 Ile Gly Trp Leu Gl n Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 120 <212> PRT <213 > Artificial Sequence <220> <221> DOMAIN <223> hu2F7 HCVR <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu2F7 LCVR <400> 18 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Ser Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2G6 HC <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu G lu Trp Val 35 40 45 Ser Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 20 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2G6 LC <400> 20 Asp Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys AlaPro Lys Ala Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 21 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2F7 HC <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 22 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2F7 LC <400> 22 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Ser Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Se r 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 < 210> 23 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR1 <400> 23 Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn 1 5 10 <210> 24 <211 > 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400> 24 Gly Ile Tyr Pro Asn Ser Gly Asn Ile Glu Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR3 <400> 25 Asp Ser Arg Phe Ser Tyr 1 5 <210> 26 <211> 11 <212> PRT < 213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 <400> 26 Lys Ala Ser Gln Asp Val Arg Thr Ala Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <22 3> LCDR2 <400> 27 Ser Thr Ser Tyr Arg Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR3 <400> 28 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR1 <400> 29 Gly Asp Thr Phe Thr Thr Tyr 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR2 <400> 30 Tyr Leu Asn Ser Gly Ser 1 5 <210> 31 <211> 6 <212 > PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HCDR3 <400> 31 Asp Ser Arg Phe Ser Tyr 1 5 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR1 <400> 32 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR2 <400> 33 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> LCDR 3 <400> 34 Gln Gln His Tyr Asn Thr Pro Leu Thr 1 5 <210> 35 <211> 115 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu2F8 HCVR <400> 35 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu2F8 LCVR <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 115 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu1C9 HCVR <400> 37 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Thr Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile Tyr Leu Asn Arg Gly Ser Ser Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 38 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu1C9 LCVR <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 39 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2F8 HC <400> 39 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gin Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 20 5 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 40 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu2F8 LC <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 P he Asn Arg Gly Glu Cys 210 <210> 41 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu1C9HC <400> 41 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Thr Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile Tyr Leu Asn Arg Gly Ser Ser Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 2 95 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 4 40 445 <210> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu1C9 LC <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Optimized HCDR1 <400> 43 Gly Tyr Thr Phe Thr Ser Ser Trp Met Asn 1 5 10 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Optimized HCDR2 <400> 44 Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Optimized HCDR2 <400> 45 Tyr Leu Asn Arg Gly Ser 1 5 <210> 46 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2G6 HCVR <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 47 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2G6 LCVR <400> 47 Asp Ile Arg Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly 1 5 10 15 Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Ala Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Ph e Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 48 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2F7 HCVR <400 > 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 49 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2F7 LCVR <400> 49 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg V al Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Ser Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 50 <211> 115 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2F8 HCVR <400> 50 Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser 20 25 30 Trp Met Asn Trp Ala Lys Leu Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Tyr Pro Asn Ser Gly Asn Ile Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 51 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 2F8 LCVR <400> 51 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Ser Ser Thr Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Ile Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 52 <211> 115 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 1C9 HCVR <400> 52 Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asp Thr Phe Thr Thr Tyr 20 25 30 Trp Met Asn Tr p Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Tyr Leu Asn Ser Gly Ser Ser Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Ser Val Asp Thr Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 53 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> 1C9 LCVR <400> 53 Asp Ile Val Met Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Glu Leu Leu Ile 35 40 45 Ser Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu L ys 100 105 <210> 54 <211> 330 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> IgG1 C <400> 54 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 2 95 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 55 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ig kappa C<400> 55 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims (41)

An antibody-drug conjugate represented by the following formula (A) or a pharmaceutically acceptable salt or solvate thereof:
[Formula A]

In the above formula,
D is a cytotoxic drug;
L ₁ and L ₂ are linker units;
y is a number from 1 to 20;
Ab is a B7-H4 antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region;
The antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30 and comprising at least one HCDR set forth in a sequence selected from the group consisting of SEQ ID NO: 31;
The antibody light chain variable region comprises SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 33 and one or more LCDRs set forth in a sequence selected from the group consisting of SEQ ID NO:34. According to claim 1,
An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody heavy chain variable region of Ab comprises any one CDR selected from the group consisting of the following (1) to (4):
(1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5;
(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11;
(3) HCDR1 set forth in SEQ ID NO:23, HCDR2 set forth in SEQ ID NO:24 and HCDR3 set forth in SEQ ID NO:25; and
(4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31. According to claim 1,
An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody light chain variable region of Ab comprises any one CDR selected from the group consisting of the following (1) to (4):
(1) LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8;
(2) LCDR1 set forth in SEQ ID NO: 12, LCDR2 set forth in SEQ ID NO: 13 and LCDR3 set forth in SEQ ID NO: 14;
(3) LCDR1 set forth in SEQ ID NO:26, LCDR2 set forth in SEQ ID NO:27 and LCDR3 set forth in SEQ ID NO:28; and
(4) LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34. According to claim 1,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody heavy chain variable region and the antibody light chain variable region of Ab comprise any one CDR selected from the group consisting of the following (1) to (4):
(1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5; and
LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7 and LCDR3 set forth in SEQ ID NO: 8;
(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11; and
LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13 and LCDR3 shown in SEQ ID NO: 14;
(3) HCDR1 set forth in SEQ ID NO:23, HCDR2 set forth in SEQ ID NO:24 and HCDR3 set forth in SEQ ID NO:25; and
LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28; and
(4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30 and HCDR3 set forth in SEQ ID NO: 31; and
LCDR1 set forth in SEQ ID NO:32, LCDR2 set forth in SEQ ID NO:33 and LCDR3 set forth in SEQ ID NO:34. 5. The method according to any one of claims 1 to 4,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, or a humanized antibody or fragment thereof. 6. The method according to any one of claims 1 to 5,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein Ab also comprises any one of (a), (b) and (c) or a combination thereof:
(a) a light chain framework region and a heavy chain framework region derived from human germline light and heavy chain sequences or mutant sequences thereof;
(b) a heavy chain constant region derived from human IgG1 or variant thereof, IgG2 or variant thereof, IgG3 or variant thereof, or IgG4 or variant thereof, preferably a heavy chain constant region derived from human IgG1, IgG2 or IgG4, more preferably is a heavy chain constant region of IgG1 with enhanced ADCC toxicity after amino acid mutation, most preferably the heavy chain constant region set forth in SEQ ID NO: 54; and
(c) a light chain constant region derived from a human κ chain, a λ chain or a variant thereof, preferably a light chain constant region derived from a human κ chain, more preferably the light chain constant region set forth in SEQ ID NO:55. 7. The method according to any one of claims 1 to 6,
Ab is at least 70%, 75%, 80%, 85 with a light chain variable region of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36 or SEQ ID NO: 38, or SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36 or SEQ ID NO: 38 An antibody-drug conjugate comprising a light chain variable region having %, 90%, 95% or 99% homology, or a pharmaceutically acceptable salt or solvate thereof. 8. The method according to any one of claims 1 to 7,
Ab is at least 70%, 75%, 80%, 85 with the heavy chain variable region of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 37, or SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35 or SEQ ID NO: 37 An antibody-drug conjugate comprising a heavy chain variable region having %, 90%, 95% or 99% homology, or a pharmaceutically acceptable salt or solvate thereof. 9. The method according to any one of claims 1 to 8,
The light chain of the Ab is at least 80%, 85%, 90%, 95% or An antibody-drug conjugate selected from the group consisting of a full-length light chain having 99% homology, or a pharmaceutically acceptable salt or solvate thereof. 10. The method according to any one of claims 1 to 9,
The heavy chain of the Ab is at least 80%, 85%, 90%, 95% or An antibody-drug conjugate selected from the group consisting of a full-length heavy chain having 99% homology, or a pharmaceutically acceptable salt or solvate thereof. 11. The method according to any one of claims 1 to 10,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy and light chain variable regions of Ab are any one selected from the group consisting of:
(1) a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16;
(2) a heavy chain variable region set forth in SEQ ID NO: 17 and a light chain variable region set forth in SEQ ID NO: 18;
(3) a heavy chain variable region set forth in SEQ ID NO: 35 and a light chain variable region set forth in SEQ ID NO: 36; and
(4) a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 38. 12. The method of claim 11,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is any one selected from the group consisting of:
(1) a light chain set forth in SEQ ID NO: 20 and a heavy chain set forth in SEQ ID NO: 19;
(2) a light chain set forth in SEQ ID NO: 22 and a heavy chain set forth in SEQ ID NO: 21;
(3) a light chain set forth in SEQ ID NO:40 and a heavy chain set forth in SEQ ID NO:39; and
(4) a light chain set forth in SEQ ID NO: 42 and a heavy chain set forth in SEQ ID NO: 41. 13. The method according to any one of claims 1 to 12,
the antigen-binding fragment is selected from the group consisting of an antigen-binding fragment of a peptide comprising Fab, Fab', F(ab') ₂ , a single chain antibody, a dimerized V region, a disulfide bond stabilized V region, and a CDR An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof. 14. The method according to any one of claims 1 to 13,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutic agents, antibiotics, radioisotopes and nucleases. 15. The method according to any one of claims 1 to 14,
cytotoxic drugs
tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division;
preferably DM1, DM3, DM4, SN-38, MMAF and MMAE;
More preferably the tubulin inhibitors SN-38, MMAE and MMAF
An antibody-drug conjugate selected from the group consisting of, or a pharmaceutically acceptable salt or solvate thereof. 15. The method according to any one of claims 1 to 14,
An antibody-drug conjugate wherein the cytotoxic drug is selected from camptothecin derivatives, preferably Exatecan, or a pharmaceutically acceptable salt or solvate thereof:

16. The method according to any one of claims 1 to 15,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from the group consisting of:

According to claim 1,
An antibody-drug conjugate represented by Formula I, or a pharmaceutically acceptable salt or solvate thereof:
[Formula I]

In the above formula,
L ₁ and L ₂ are linker units;
y is a number selected from 1 to 8, preferably a number selected from 2 to 4;
Ab is the B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. According to claim 1,
An antibody-drug conjugate represented by Formula II, or a pharmaceutically acceptable salt or solvate thereof:
[Formula II]

In the above formula,
L ₁ and L ₂ are linker units;
y is a number selected from 1 to 8, preferably a number selected from 2 to 4;
Ab is the B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. According to claim 1,
An antibody-drug conjugate represented by Formula III, or a pharmaceutically acceptable salt or solvate thereof:
[Formula III]

In the above formula,
L ₁ and L ₂ are linker units;
y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8, even more preferably a number from 6 to 8, most preferably 8;
Ab is the B7-H4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. 21. The method according to any one of claims 18 to 20,
L ₁ is an antibody-drug conjugate represented by Formula B: or a pharmaceutically acceptable salt or solvate thereof:
[Formula B]

In the above formula,
M ₁ is —CR ₁ R ₂ —;
R ₁ and R ₂ are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino;
n is an integer from 0 to 5, preferably 1, 2 or 3. 21. The method according to any one of claims 18 to 20,
L ₂ is an antibody-drug conjugate represented by formula (C): or a pharmaceutically acceptable salt or solvate thereof:
[Formula C]

In the above formula,
M ₂ is —CR ₄ R ₅ —;
R ₃ is selected from the group consisting of hydrogen, halogen, hydroxyl, amino, alkyl, alkoxyl and cycloalkyl;
R ₄ and R ₅ are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino;
m is an integer from 0 to 5, preferably 1, 2 or 3. 21. The method according to any one of claims 18 to 20,
L ₂ is an antibody-drug conjugate represented by formula (D): or a pharmaceutically acceptable salt or solvate thereof:
[Formula D]
-K ¹ -K ² -K ³ -K ⁴ -
In the above formula,
K ¹ is

and s is an integer from 2 to 8;
K ² is -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ CH ₂ C(O)-, -NR ¹ (CH ₂ CH ₂ O) _p CH ₂ C(O)-, -S(CH ₂ ) _p selected from the group consisting of C(O)- and a single bond, p is an integer from 1 to 20, preferably from 1 to 6;
R ₁ is selected from the group consisting of hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;
K ³ is a tetrapeptide residue, preferably the tetrapeptide residue is a peptide residue formed by an amino acid selected from the group consisting of at least two of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate and aspartate; more preferably the tetrapeptide residue GGFG;
K ⁴ is —NR ² (CR ³ R ⁴ ) _t —, and R ² , R ³ and R ⁴ are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxy hydroxyalkyl; t is 1 or 2. 24. The method of claim 23,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the K ¹ terminus of the linker unit -L ₂ - is linked to Ab and the K ⁴ terminus is linked to L ₁ . 21. The method according to any one of claims 18 to 20,
L ₁ is -O-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-, -O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -, -O-CR ⁵ R ⁶ - , -NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)- and -S-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)- and ;
R ^a and R ^b are each independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl;
R ⁵ is haloalkyl or cycloalkyl,
R ⁶ is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl,
R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl;
m is 0, 1, 2, 3 or 4
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof. 26. The method of claim 25,
An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the O terminus of L ₁ is linked to a linker unit L ₂ . 26. The method of claim 25,
L ₁ is an antibody-drug conjugate represented by Formula E: or a pharmaceutically acceptable salt or solvate thereof:
[Formula E]

In the above formula,
R ⁵ is selected from the group consisting of haloalkyl and cycloalkyl,
R ⁶ is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl,
R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl;
preferably
R ⁵ is selected from the group consisting of C _1-6 haloalkyl and C _3-6 cycloalkyl,
R ⁶ is selected from the group consisting of hydrogen, C _1-6 haloalkyl and C _3-6 cycloalkyl,
R ⁵ and R ⁶ and the carbon atom to which they are connected form a C _3-6 cycloalkyl;
m is an integer from 0 to 4;
More preferably, formula E is selected from the group consisting of the following substituents:

28. The method according to any one of claims 1 to 27,
-L ₂ -L ₁ -An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, as shown in the structure below:

In the above formula,
K ² is a bond;
K ³ is the tetrapeptide residue GGFG;
R ⁵ is selected from the group consisting of haloalkyl and C _3-6 cycloalkyl,
R ⁶ is selected from the group consisting of hydrogen, haloalkyl and C _3-6 cycloalkyl,
R ⁵ and R ⁶ and the carbon atom to which they are connected form a C _3-6 cycloalkyl;
R ² , R ³ and R ⁴ are each independently hydrogen or alkyl;
s is an integer from 2 to 8;
m is an integer from 0 to 4;
Preferably, said -L ₂ -L ₁ - is selected from the group consisting of:

. 13. The method according to any one of claims 1 to 12,
An antibody-drug conjugate represented by Formula IV, or a pharmaceutically acceptable salt or solvate thereof:
[Formula IV]

In the above formula,
W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-cycloalkyl and linear heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl is 1 to selected from the group consisting of N, O and S contains 3 heteroatoms, wherein said C _1-8 alkyl, cycloalkyl and linear heteroalkyl consist of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl. optionally further substituted with one or more substituents selected from the group;
K ² is -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, -S(CH ₂ ) _p1 C(O)— and a bond, R ¹ is selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, p ₁ is an integer from 1 to 20;
K ³ is a peptide residue consisting of 2 to 7 amino acids, which amino acids may be substituted or unsubstituted; When substituted, said substituent may be substituted at any available point of attachment, said substituent being from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl. at least one independently selected from;
R ² is independently selected from the group consisting of a hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
R ³ and R ⁴ are each independently selected from the group consisting of a hydrogen atom, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
R ⁵ is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;
R ⁶ is selected from the group consisting of a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;
R ⁵ and R ⁶ and the carbon atom to which they are connected form a cycloalkyl or heterocyclyl;
m is an integer from 0 to 4;
y is 1 to 10, y is a decimal or an integer;
Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof. 19. The method of claim 18,
An antibody-drug conjugate represented by Formula IA, or a pharmaceutically acceptable salt or solvate thereof:
[Formula IA]

. 19. The method of claim 18,
An antibody-drug conjugate represented by Formula IB, or a pharmaceutically acceptable salt or solvate thereof:
[Formula IB]

. 20. The method of claim 19,
An antibody-drug conjugate represented by Formula II-A, or a pharmaceutically acceptable salt or solvate thereof:
[Formula II-A]

. 20. The method of claim 19,
An antibody-drug conjugate represented by Formula II-B, or a pharmaceutically acceptable salt or solvate thereof:
[Formula II-B]

. 30. The method of claim 29,
It is presented in Formula IV-A below:
[Formula IV-A]

,
Preferably, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as shown below:

. 35. The method of any one of claims 1 to 34,
An antibody-drug conjugate selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt or solvate thereof:

In the above formula,
y is selected from 2 to 10, preferably 4 to 8, more preferably 6 to 8, still more preferably 7 to 8, and most preferably 8. A method for preparing an antibody-drug conjugate shown in Formula IV or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of: performing a coupling reaction with a compound shown in Formula F when Ab is reduced to obtain the formula Methods of preparing the compounds set forth in IV:

In the above formula,
Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof;
W, K ² , K ³ , R ² to R ⁶ , m and y are as defined in claim 29 . 37. The method of claim 36,
A method for preparing the compound represented by Formula F, wherein the compound represented by Formula F-1 below, or a tautomer, mesomemer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:
[Formula F-1]

In the above formula,
K ² , K ³ , R ² to R ⁶ , s and m are as defined in claim 28 . A compound represented by Formula F or Formula F-1 selected from the group consisting of:

. 36. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 35, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable carrier. 40. The antibody-drug conjugate according to any one of claims 1 to 35, or a pharmaceutically acceptable salt or solvate thereof, or according to claim 39, in the manufacture of a medicament for the treatment of a disease associated with human B7-H4. Use of a pharmaceutical composition. 41. The method of claim 40,
Use for the manufacture of a medicament for the treatment of a cancer having high B7-H4 expression, wherein the cancer is astroblastoma of the human brain, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft sarcoma, astrocytoma, bladder cancer, Bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophore cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative bovine Atopic cell tumor, ependymoma, Ewing's sarcoma, extraosseous mucinous chondrosarcoma, osseous incomplete fibrosis, fibrous dysplasia of bone, gallbladder or cholangiocarcinoma, gastric cancer, gestational villous disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell Tumor, Kaposi's sarcoma, renal cancer, leukemia, liposarcoma/malignant lipoma tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovary Cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, childhood cancer, peripheral schwannoma, pheocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis, rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous A use selected from the group consisting of cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thyroid metastasis and uterine cancer.

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